Advances in the biological, biomedical and pharmaceutical sciences have accelerated the pace of research and diagnostics unparalleled in the past. With whole genome sequences becoming quickly and successively available, the assembly of large libraries of small molecules, and the ability to move pharmaceutical development, clinical diagnostic tests and basic research from a reductionist to a whole system approach demands assays that facilitate high throughput analyses. Molecules no longer need to be singly analyzed for their effects on a lone process; instead, the effects of many molecules on several biological systems can be studied simultaneously—if appropriate, fast, reliable, and accurate assays are available.
Preferred bioassays that assist in evaluating cellular health are those that detect and quantify adenosine triphosphate (ATP). Hydrolysis of ATP powers many of a cell's biochemical processes. Healthy, viable cells are rich in ATP; dead or dying cells are ATP-poor.
Efficient, reliable and accurate assays for cell viability can be used to rapidly discover cytotoxic agents or cell proliferation agents and determine the cytotoxic effect or cell proliferation effect of agents on cells. Cancer pharmaceutical research often endeavors to identify compounds that selectively kill quickly-dividing cells—a primary characteristic of cancer cells. While some effective anti-cancer cytotoxic compounds have been identified; innumerable potentially more valuable compounds await identification. High throughput screens of compound libraries, coupled with efficient cell viability assays, can swiftly identify such compounds. In some systems of the body, controlled cell death is crucial for appropriate function. For example, immune system development—a continual process—depends on apoptosis (programmed cell death). The discovery of drugs to treat immuno-related dysfunctions often depends on determining cell viability. The efficacy of a candidate compound on cell viability can be assayed by detecting ATP, since ATP production is only realized in metabolically active (live) cells and residual ATP in a cell is degraded upon cell death, particularly quickly in non-apoptotic (necrotic) cell death. Assay systems that not only facilitate the evaluation of a substance on cell viability, but also permit high throughput screens that can rapidly test thousands of compounds, streamline new drug discovery.
In clinical settings, diagnostic tests on large numbers of samples are facilitated when simple, accurate and safe assays are used. Disease treatments can then be more readily determined and instituted.
With the availability of whole genome sequences, the identification of gene products that affect ATP production, either indirectly or directly, is made possible, and high throughput screens to identify such proteins are facilitated by simple, fast, accurate and reliable ATP assays.
ATP assays are valuable for innumerable types of measurements for which it is important to determine the presence or absence of microbes or to determine the amount of microbial contamination present, e.g., determining microbial contamination of end products, hygiene monitoring, effectiveness of biocides, success of biological waste treatment processes, and the like.
ATP assays depend on reporter molecules or labels to qualitatively or quantitatively monitor ATP levels. Reporter molecules or labels in such assay systems have included radioactive isotopes, fluorescent agents, and enzymes, including light-generating enzymes such as luciferase. Desirable characteristics of any reporter molecule systems include safe, quick and reliable application and detection. Luminescent systems are among the most desirable since they are exceptionally safe and sensitive.
Light-generating enzymes have been isolated from certain bacteria, protozoa, coelenterates, mollusks, fish, millipedes, flies, fungi, worms, and crustaceans. Those enzymes isolated from beetles, particularly the fireflies of the genera Photinus, Photuris, and Luciola, and from click beetles of genus Pyrophorus, have found widespread use in reporter systems. In many of these organisms, enzymes such as luciferases catalyze oxido-reductions in which the free energy change excites a substrate molecule to a high-energy state. When the excited molecule returns to the ground state, visible light is emitted, i.e. “bioluminescence” or “luminescence.” Among the assay systems in which bioluminescence has been employed to monitor or measure ATP are those in which the activity of an ATP-dependent bioluminescent enzyme, e.g. a beetle luciferase, is exploited.
When luciferase is combined with a sample for the purpose of detecting ATP, it is typically desirable to inhibit ATPases endogenous to the sample as well as enzymes that generate ATP, thus assuring that the ATP detected corresponds to the actual amount of ATP in a sample at a desired time. Many ATPase inhibitors are known, including detergents, especially detergents that are positively charged. However, most ATPase inhibitors are effective in not only eliminating ATPase function endogenous to the sample (e.g., a cell or cell population), but also ATPases that may be used as the reporter molecule, such as luciferase. Additionally, to counter ATP production, inhibitors of enzymes that phosphorylate, such as kinases, are desirable. However, these inhibitors, such as sodium fluoride (NaF), might also affect luciferase function. A challenge to improving ATP detection in a sample using luciferase depends on methods or compositions that substantially decrease or eliminate ATPase activity and ATP-generating activity endogenous to the sample, thereby stabilizing the amount of ATP present in the sample to that present when the composition is added, without confounding luciferase function.
There are multiple variations of cellular ATP detection methods currently used, all of which act in a stepwise manner. Some such methods first lyse the cells and inactivate the ATPase activity endogenous to the sample (e.g., by increasing sample pH), then neutralize the ATPase inhibitor, thereby converting the environment of the sample to one favorable to luciferase activity prior to adding the luciferase and detecting luminescence. Other such methods combine the neutralization of the ATPase inhibitor with the addition of luciferase. There are no ATP detection systems that provide a composition or method capable of inactivating endogenous ATPase activity and detecting luciferase activity in the same environmental milieu. Therefore, current assays that use luminescence to detect ATP are handicapped by the need for successive, time-consuming steps.
The present invention provides compositions with properties of enhanced stability comprising a luciferase and one or more ATPase inhibitors and further provides methods using these novel compositions to detect ATP in a sample by reducing the steps of cell lysis, endogenous ATPase inhibition, and substrate and luciferase addition to a single step that is then followed by detection of luminescence. Because embodiments of the invention significantly reduce the time and effort of luciferase-mediated detections of ATP by eliminating the need to neutralize ATPase inhibitor activity before adding luciferase, high throughput assays can finally be efficiently realized.